Detection of degradation products of canine NT-proBNP

ABSTRACT

A method for determining the amount of NT-proBNP in blood samples from animals. The method includes detecting degradation products of NT-proBNP by various methods, including using antibodies, kits and device.

CROSS REFERENCE TO RELATED APPLICATION

This application claims benefit of U.S. Provisional Application Ser. No.61/370,712 filed Aug. 4, 2010, which is incorporated herein by referencein its entirety.

SEQUENCE LISTING STATEMENT

The sequence listing is filed in this application in electronic formatonly and is incorporated by reference herein. The sequence listing textfile “09-1182-US_SEQ.txt” was created on Aug. 3, 2011, and is 13,087bytes in size.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is related to the detection of NT-proBNP in biologicalsamples from canines.

2. Description of Related Art

Heart diseases affect all non-human animals. Heart diseases generallyinvolve the cardiac valves and the cardiac muscle. Because the heart iscapable of compensating for functional impairment by working harder,heart diseases in most cases remain hidden, with the consequence thatthe health of the heart will deteriorate due to the increased workloadon the heart. The symptoms resulting from heart diseases, such asfatigue, circulatory insufficiency, and languor, can usually berecognized when the animal's heart is no longer able to compensate forthe a particular functional impairment. At this stage, the heart diseasehas progressed to the point that curing the disease is no longerpossible.

While chronic cardiac valve and cardiac muscle changes are usuallyincurable, the use of medicaments can slow disease progression.Therefore, an early diagnosis for heart diseases is beneficial.Routinely, physical methods are used for this purpose, such asauscultation of the heart sounds, the recording of an electrocardiogram,and X-ray and ultrasonic examinations. These examination methods havethe disadvantage that they are carried out only when already visible oraudible defects of the heart are recognized. Furthermore, physicalexamination methods require suitable and generally expensive devices inorder to carry out a respective diagnosis.

In many heart diseases, such as, e.g., heart decompensation and dilatedcardiomyopathy, a peptide hormone—the so-called brain natriureticpeptide (BNP)—is secreted by heart muscle tissue. Since this hormone isproduced in the heart and is increasingly produced in case of overstressand congestion, determining the BNP level in blood is a suitable meansfor evaluating cardiac insufficiency.

BNP as well as other natriuretic peptides play an important part inregulating water balance and blood pressure. If the cardiac wall isdilated, it secrets BNP in increasing amounts, which in turn causes anexcretion of sodium and liquid via the kidneys and dilation of the bloodvessels. These factors can lower the blood pressure and the fillinglevel of the heart. BNP is synthesized by the cells of the cardiacmuscle as proBNP, which is cleaved into N-terminal proBNP (NT-proBNP)and BNP. Both parts of the polypeptide are delivered to the blood andcan be detected therein.

The utility of both BNP and NT-proBNP as makers for cardiac disease inveterinary patients (e.g., dogs and cats) has been demonstrated innumerous studies. For instance, BNP and NT-proBNP assays have been shownto be effective as a diagnostic test for dogs, as illustrated by twostudies, which report remarkably similar sensitivity and specificity(85% and 82% respectively) for differentiating the cause of clinicalsigns that may be attributable to cardiac disease in dogs. See Oyama MA, et al., “Assessment of serum N-terminal pro-B-type natriureticpeptide concentration for differentiation of congestive heart failurefrom primary respiratory tract disease as the cause of respiratory signsin dogs”, Journal of American Veterinary Medical Association (December2009); Boswood et al., “The diagnostic accuracy of different natriureticpeptides in the investigation of canine cardiac disease”, JSAP 2007 1-7.In cats, the clinical challenge is different, as the most common cardiacdisease in cats is hypertrophic cardiomyopathy. This disease remainsoccult or ‘silent’ with very few clinical signs that are appreciable tothe pet owner until the disease is very advanced.

A number of immunoassays for the detection of NT-proBNP are known. Theseassays use polyclonal or monoclonal antibodies specific for epitopeswithin various regions of the peptide. These methods, however, aresubject to variability because NT-proBNP is further processed ex vivo byvarious proteases in the blood serum and plasma. Therefore, immunoassaysfor NT-proBNP are inherently vulnerable to inconsistency due to the exvivo degradation of the protein over time. Therefore, samples to betested for NT-proBNP are typically refrigerated and efforts are made toconduct sample analysis as quickly as possible following taking of thesample.

Accordingly, the inventors have identified a need in the art for amethod of determining NT-proBNP that can measure NT-proBNP withoutregard to when a sample was taken and without cumbersome handlingrequirements for the sample.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to a method for determining theamount of NT-proBNP in a sample from a canine, comprising determiningthe amount of a polypeptide selected from a sequence consistingessentially of one of GRSPASEASEASEASGLWAVQ (SED ID NO: 22) andSHSPAEAPEAGGTPRGVLAPHDSVLQ (SEQ ID NO: 30) in the sample, andcorrelating the amount of the polypeptide in the sample to the amount ofNT-proBNP in the sample.

In another aspect, the invention is directed to a method for determiningthe amount of NT-proBNP in a sample from a canine, the method comprisingcontacting the sample with a first monoclonal antibody that specificallybinds a polypeptide selected from a sequence consisting essentially ofone of SEQ ID NO: 22 and SEQ ID NO: 30 or both; determining the amountof binding of at least a fragment of the NT-proBNP in the sample to thefirst monoclonal antibody; and associating the amount of binding to theamount of NT-proBNP in the sample.

In a further aspect, the invention is directed to a method fordetermining the presence or amount of NT-proBNP in a biological samplecomprising forming a mixture of the sample with a first monoclonalantibody that specifically binds a fragment of NT-proBNP that is stablein the sample for at least 96 hours; allowing at least a fragment of theNT-proBNP in the sample and the monoclonal antibody to form a complex;simultaneously or sequentially contacting the mixture with a secondmonoclonal antibody that binds to NT-proBNP, wherein the firstmonoclonal antibody is immobilized on a solid phase and the secondmonoclonal antibody is conjugated to a label, or the first antibody isconjugated to a label and the second antibody is immobilized on thesolid phase; and detecting the presence or amount of the label on thesolid phase.

In yet another aspect, the invention is directed to a method fordetermining the presence or amount of NT-proBNP in a biological samplecomprising detecting a degradation product of NT-proBNP in the sample,wherein the degradation product is a fragment of NT-proBNP that isstable in the sample for at least 96 hours.

In another aspect, the invention is directed to a kit for determiningthe presence, absence or level of canine NT-proBNP in a samplecomprising a solid support; a first antibody, the first antibody beingspecific for a first epitope on a fragment of canine NT-proBNP, whereinthe fragment is less than the full length of canine NT-proBNP and isstable in canine serum at room temperature for at least 96 hours afterobtaining the sample; and a second antibody being specific for a secondepitope on the fragment, wherein the second epitope is different thanthe first epitope.

In another aspect, the invention is directed to an immunoassay devicecomprising a solid support having a first antibody bound thereto, thefirst antibody being specific for a first epitope on a fragment ofcanine NT-proBNP; stability in canine serum at room temperature for atleast 96 hours after obtaining the sample from the canine; and a secondantibody being specific for a second epitope on the fragment of canineNT-proBNP, the second epitope being different than the first epitope.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides an alignment of BNP polypeptide sequences from human,canine and feline (SEQ ID NOS 1-3).

FIG. 2 shows the results of the degradation of NT-proBNP in plasma.

FIGS. 3-6 show the relative amounts of NT-proBNP polypeptide fragmentsin plasma samples after 0, 24, 96 and 192 hours of spiking withrecombinant NT-proBNP.

FIG. 7 shows the results of a sandwich assay using a monoclonal antibodyspecific for a stable fragment of NT-proBNP of the course of 96 hours.

FIG. 8 shows the results of a sandwich assay using a monoclonal antibodyspecific for a stable fragment of NT-proBNP over the course of 96 hours.

DETAILED DESCRIPTION

Degradation products of NT-proBNP can be determined to provide anaccurate and reliable measurement of the original amount of NT-proBNP ina fresh blood sample. Because the stable degradation products, and notthe NT-proBNP itself, are determined, the urgency to test the sampleimmediately or to refrigerate the sample prior to testing is eliminated.

There are several ways to assay a sample (e.g. whole blood, serum orplasma) for NT-proBNP or its degradation products. Mass Spectrometry,for example, can be used to look for a particular peptide sequence (e.g.full length NT-proBNP or a portion thereof). Immunoassay techniques canalso be used to directly or indirectly detect full length NT-proBNP or aportion thereof. In a direct assay format, monoclonal antibodies,polyclonal antibodies or combinations thereof can be used to capture anddetect full length or fragments of NT-proBNP (a.k.a. “sandwich” assayswhere one antibody captures the target analyte and a second, labeledantibody detects the captured analyte).

When assaying for NT-proBNP fragments in a sandwich assay, it isimportant to ensure the antibody pair is specific for epitopes thatexist on the target, i.e. the assay will miss the fragment if one ormore of the epitopes are cleaved from the original peptide or otherwisebecome compromised or inactivated. For example, assume the line, below,is full length canine |NT-proBNP|, wherein, over time, the peptidedegrades into portions {A}, [B] and <C>, ([B] and <C> being degradationproducts of A):

An antibody pair, one directed to an epitope on [B] and the other to anepitope on <C>, will work in a sandwich assay for {A} (or the fulllength peptide) as long as {A} does not split into [B] and <C>. If {A}splits into [B] and <C> a working assay for this section of the fullpeptide would need to have an antibody pair directed towards activeepitopes on either [B] or <C>.

Alternatively, in a competition immunoassay format, only one epitope ofa target fragment need be identified and targeted with either amonoclonal or polyclonal antibody. In this format, if thefragment/epitope is present, it will bind with the antibody and“compete” with a detection reagent that would have otherwise bound tothe antibody.

Preferred target NT-proBNP fragments (discussed in detail, below) arethose that exist in detectable concentrations over time. By determiningthose fragments that are stable over time one can choose MassSpectrometry parameters or antibodies to detect and determine theconcentration of the stable fragment. Once one determines theconcentration of a stable fragment, the original amount of NT-proBNP inthe sample can be determined.

Stable fragments NT-proBNP can also be used to purify polyclonalantibodies from sera (e.g., from chickens, goats, sheep and/or donkeysthat have been immunized with all or a portion of the NT-proBNPsequence).

In order to assess the extent of heart damage or disease, determinationof the quantity of NT-proBNP in a sample is helpful. Quantitativedetermination of the degradation products of NT-proBNP can beaccomplished by several methods. Once the amount of NT-proBNP or one ormore of its degradation products (the analyte) in a sample isdetermined, the amount NT-proBNP in the sample, prior to degradation,can be determined.

Accordingly, in one aspect the invention is directed to determining theamount of NT-proBNP in a sample from a canine, wherein the methodincludes determining the amount of a degradation product of NT-proBNP(i.e., a polypeptide fragment of native canine NT-proBNP). The amount ofNT-proBNP in the sample can be used as a measurement of cardiac diseasein the animal.

Substantial homology between species exists for BNP and NT-proBNP. Forexample, FIG. 1 provides an alignment of BNP polypeptide sequences fromhuman, canine and feline (SEQ ID NOs. 1-3). As described above, BNP isprocessed prior to secretion from muscle cells as the N-terminal pro-BNP(NT-proBNP) and BNP.

Recombinant Canine NT-proBNP (a.k.a. rNT-proBNP or synthetic NT-proBNP)has been reported to have the following polypeptide sequence (SEQ ID NO:4):

HPLGGRSPAS EASEASEASG LWAVQELLGR LKDAVSELQAEQLALEPLHR SHSPAEAPEA GGTPRGVLAP HDSVLQALRIt is noted that all experiments performed herein were carried out usingrecombinant Canine NT-proBNP.

This peptide lacks the three C-terminal amino acids of the nativesequence as a result of the expression vector used.

As shown in FIG. 2, canine NT-proBNP is degraded (cleaved in at leastone location) nearly 80% after 24 hours ex vivo in plasma. Because thepeptide is quickly degraded, epitopes specific for various antibodiesmay no longer be available for binding to the antibodies. In addition,when an assay relies upon antibody pairs that bind to the samepolypeptide, cleavage through degradation of the polypeptide in theregion between the two epitopes recognized by the antibodies results inthe antibodies not longer being able to associate and provide a signalthat is indicative of the association.

The degradation products of NT-proBNP are numerous. While all of theserum degradation products have yet to be identified, expecteddegradation products from a number of known proteases can be determined.For example, Table 1 shows a the expected fragments from the digestionof canine NT-proBNP with Trypsin as predicted by the Peptide Cutterapplication available at the ExPASy (Expert Protein Analysis System)proteomics server of the Swiss Institute of Bioinformatics (SIB).

TABLE 1 Peptide Peptide Position of Resulting peptide sequence lengthmass Cleavage cleavage site (see explanations) [aa] [Da] probability 6HPLGGR 6 635.724 100% (SEQ ID NO: 5) 30 SPASEASEASEASGLWAVQELLGR 242445.625 100% (SEQ ID NO: 6) 32 LK 2 259.349 86.4%  50DAVSELQAEQLALEPLHR 18 2019.242 100% (SEQ ID NO: 7) 65 SHSPAEAPEAGGTPR 151463.527 100% (SEQ ID NO: 8) 79 GVLAPHDSVLQALR 14 1475.711 —(SEQ ID NO: 9)

As another example, Table 2 shows the expected fragments from digestionof canine NT-proBNP Proteinase K as predicted by the ExPASy PeptideCutter application.

TABLE 2 Position of Peptide Peptide cleavage length mass siteResulting peptide sequence [aa] [Da] 3 HPL 3 365.432 9 GGRSPA 6 543.580(SEQ ID NO: 10) 12 SEA 3 305.288 15 SEA 3 305.288 18 SEA 3 305.288 21SGL 3 275.305 22 W 1 204.228 23 A 1  89.094 24 V 1 117.148 27 QEL 3388.421 28 L 1 131.175 31 GRL 3 344.414 34 KDA 3 332.357 35 V 1 117.14838 SEL 3 347.368 40 QA 2 217.225 43 EQL 3 388.421 44 A 1  89.094 45 L 1131.175 48 EPL 3 357.407 55 HRSHSPA 7 790.837 (SEQ ID NO: 11) 57 EA 2218.210 60 PEA 3 315.326 63 GGT 3 233.224 67 PRGV 4 427.504(SEQ ID NO: 12) 68 L 1 131.175 69 A 1  89.094 74 PHDSV 5 553.572(SEQ ID NO: 13) 75 L 1 131.175 77 QA 2 217.225 78 L 1 131.175 79 R 1174.203

For nomenclature purposes, the canine NT-proBNP sequence can be looselydivided into three regions: N-terminal region (usually including aminoacids 1-22); middle region (usually including amino acids 23-50), andC-Terminal region (usually including amino acids 51-79). Polypeptidesequence fragments from these regions were noted at 24 or 196 hours asshown below.

N-terminal region (24 hours):

(SEQ ID NO: 14) LGGRSPASEASEASEASGLWAVQELLGR

N-terminal region (192 hours)

(SEQ ID NO: 15) LGGRSPASEASEASEASGLWAVQ (SEQ ID NO: 16)LGGRSPASEASEASEASGLWAVQEL (SEQ ID NO: 17) LGGRSPASEASEASEASGLWAVQELL(SEQ ID NO: 14) LGGRSPASEASEASEASGLWAVQELLGR (SEQ ID NO: 19)GGRSPASEASEASEASGLWAVQ (SEQ ID NO: 20) GGRSPASEASEASEASGLWAVQELL(SEQ ID NO: 21) GGRSPASEASEASEASGLWAVQELLGRL (SEQ ID NO: 22)GRSPASEASEASEASGLWAVQ (SEQ ID NO: 23) GRSPASEASEASEASGLWAVQELLG

In the above sequences, the N-terminal -HP- from the native sequence iscleaved in all cases, -L- and then the first -G- are also cleaved overtime. Antibodies raised against peptides having the N-terminal -HP-,-HPL- and/or -HPLG- have been shown to have decreased sensitivity assamples age.

Middle region (192 hours)

(SEQ ID NO: 24) LWAVQELLGRLKDAVSELQAEQLALEPLHRSHSPAEAPEAGGTPRGVL(SEQ ID NO: 25)      ELLGRLKDAVSEL (SEQ ID NO: 26)          LKDAVSELQAEQL (SEQ ID NO: 27)                     EQLALEPL(SEQ ID NO: 28)                        ALEPL

C-terminal region (192 hours)

(SEQ ID NO: 30) SHSPAEAPEAGGTPRGVLAPHDSVLQ (SEQ ID NO: 31)               GVLAPHDSVLQ

FIGS. 3-6 show the relative amount of these fragments in the plasmasamples at 0, 24, 96 and 192 hours. The most prevalent polypeptidesafter 192 hours were GRSPASEASEASEASGLWAVQ (SEQ ID NO: 22) in theN-terminal region and SHSPAEAPEAGGTPRGVLAPHDSVLQ (SEQ ID NO: 30) in theC-terminal region.

The invention also provides antibodies that selectively bind to one ofthe NT-proBNP degradation products, as well as variants and furtherfragments thereof. As used herein, an antibody selectively binds atarget peptide when it binds the target peptide and does notsignificantly bind to unrelated proteins. An antibody is stillconsidered to selectively bind a peptide even if it also binds to otherproteins that are not substantially homologous with the target peptideso long as such proteins share homology with a fragment or domain of thepeptide target of the antibody. In this case, it would be understoodthat antibody binding to the peptide is still selective despite somedegree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′)₂, and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. In one aspect, the antibodies of the invention aremonoclonal antibodies produced by a mouse myeloma cell line. This cellline can be made by fusing a mouse myeloma cell line with the spleencells from mice that have been injected with the NT-proBNP fragmentsdescribed herein and suitable carrier proteins that are well known tothose of skill in the art.

In a preferred method of making monoclonal antibodies of the presentinvention, the selected immunogen is a combination of the two peptidesconjugated from both the N-terminus and the C-terminus. For example,with respect to two of the most preferred stable peptides: N-terminalGRSPASEASEASEASGLWAVQ (SEQ ID NO: 22) and C-terminalSHSPAEAPEAGGTPRGVLAPHDSVLQ (SEQ ID NO: 30); the N-terminal immunogen isa combination of GRSPASEASEASEASGLWAVQ-(PEG)6-Cys-KLH (SEQ ID NO: 32)and KLH-Cys-(PEG)6-GRSPASEASEASEASGLWAVQ (SEQ ID NO: 33), while theC-terminal immunogen is a combination ofSHSPAEAPEAGGTPRGVLAPHDSVLQ-(PEG)6-Cys-KLH (SEQ ID NO: 34) andKLH-Cys-(PEG)6-SHSPAEAPEAGGTPRGVLAPHDSVLQ (SEQ ID NO: 35). Usingstandard techniques, the aforementioned immunogens were used to obtainmurine antibodies that were able to detect stable canine NT-proBNPfragements in blood samples.

The antibodies can be used to isolate NT-proBNP or a fragment thereof bystandard techniques, such as affinity chromatography orimmunoprecipitation. In one aspect, the invention is directed to animmunological method for detecting the presence of an amount ofNT-proBNP in a biological sample. The invention provides a method, adevice and a kit that uses one or more canine NT-proBNP monoclonalantibodies. In another aspect, the method includes calibrators andstandards comprising one or more NT-proBNP polypeptides.

“Binding specificity” or “specific binding” refers to the substantialrecognition of a first molecule for a second molecule, for example apolypeptide and a polyclonal or monoclonal antibody, or an antibodyfragment (e.g., a Fv, single chain Fv, Fab′, or F(ab′)₂ fragment)specific for the polypeptide.

A “specific binding pair” is a set of two different molecules, where onemolecule has an area on its surface or in a cavity that specificallybinds to, and is therefore complementary to, an area on the othermolecule. “Specific binding partner” refers to one of these twocomplementarily binding molecules. “Specific binding pair” may refer toa ligand and a receptor, for example. In another example, the specificbinding pair might refer to an immunological pair, for example anantigen and antibody.

“Substantial binding” or “substantially bind” refer to an amount ofspecific binding or recognizing between molecules in an assay mixtureunder particular assay conditions. In its broadest aspect, substantialbinding relates to the difference between a first molecule's inabilityto bind or recognize a second molecule, and the first molecules abilityto bind or recognize a third molecule, such that the difference issufficient to allow a meaningful assay to be conducted distinguishingspecific binding under a particular set of assay conditions, whichincludes the relative concentrations of the molecules, and the time andtemperature of an incubation. In another aspect, one molecule issubstantially incapable of binding or recognizing another molecule in across-reactivity sense where the first molecule exhibits reactivity fora second molecule that is less than 25%, preferably less than 10%, morepreferably less than 5% of the reactivity exhibited toward a thirdmolecule under a particular set of assay conditions, which includes therelative concentration and incubation of the molecules. Specific bindingcan be tested using a number of widely known methods, e.g, animmunohistochemical assay, an enzyme-linked immunosorbent assay (ELISA),a radioimmunoassay (RIA), or a western blot assay.

A “blood sample” refers to a whole blood sample from an animal or itscomponents (e.g., serum, plasma, etc.).

A “label” is any molecule that is bound (via covalent or non-covalentmeans, alone or encapsulated) to another molecule or solid support andthat is chosen for specific characteristics that allow detection of thelabeled molecule. Generally, labels are comprised of, but are notlimited to, the following types: particulate metal andmetal-derivatives, radioisotopes, catalytic or enzyme-based reactants,chromogenic substrates and chromophores, fluorescent andchemiluminescent molecules, and phosphors. The utilization of a labelproduces a signal that may be detected by means such as detection ofelectromagnetic radiation or direct visualization, and that canoptionally be measured.

The label employed in the current invention could be, but is not limitedto: alkaline phosphatase; glucose-6-phosphate dehydrogenase (“G6PDH”);horse radish peroxidase (HRP); chemiluminescers such as isoluminol,fluorescers such as fluorescein and rhodamine compounds; ribozymes; anddyes.

The label can directly produce a signal, and therefore additionalcomponents are not required to produce a signal. Alternatively, a labelmay need additional components, such as substrates or co-enzymes, inorder to produce a signal. The suitability and use of such labels usefulfor producing a signal are discussed in U.S. Pat. No. 6,489,309, andU.S. Pat. No. 5,185,243, which are incorporated by reference herein intheir entirety. For example, a label may be conjugated to the specificbinding partner in a non-covalent fashion. Alternatively, the label maybe conjugated to the specific binding partner covalently. U.S. Pat. No.3,817,837, and U.S. Pat. No. 3,996,345, which are incorporated byreference herein in their entirety, describe in detail example ofvarious ways that a label may be non-covalently or covalently conjugatedto the specific binding partner.

“Solid phase” means a porous or non-porous water insoluble material.Such materials include a support or a surface such as the wall of areaction vessel. The support can be hydrophilic or capable of beingrendered hydrophilic and includes inorganic powders such as silica,magnesium sulfate, and alumina; natural polymeric materials,particularly cellulosic materials and materials derived from cellulose,such as fiber containing papers, e.g., filter paper, chromatographicpaper, etc.; synthetic or modified naturally occurring polymers, such asnitrocellulose, cellulose acetate, poly (vinyl chloride),polyacrylamide, cross linked dextran, agarose, polyacrylate,polyethylene, polypropylene, poly(4-methylbutene), polystyrene,polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinylbutyrate), etc.; either used by themselves or in conjunction with othermaterials; glass available as Bioglass, ceramics, metals, and the like.Natural or synthetic assemblies such as liposomes, phospholipidvesicles, and cells can also be employed.

Binding of specific binding pair members to a support or surface may beaccomplished by well-known techniques, commonly available in theliterature. See, for example “Immobilized Enzymes,” Ichiro Chibata,Halsted Press, New York (1978) and Cuatrecasas, J. Biol. Chem., 245:3059(1970). The surface can have any one of a number of shapes, such asstrip, rod, particle, including bead, and the like. In one aspect, thepolypeptides of the invention include a N-terminal cysteine residue toassist in binding the polypeptides to the solid phase.

The method of the invention can be optimized in many ways and one ofskill in the art could simultaneously adjust the sample dilutions,reagent concentrations, incubation temperatures and times used in themethod to accomplish detection of NT-proBNP.

To be useful in the detection methods of the present invention, thepolypeptides are obtained in a substantially pure form, that is,typically from about 50% w/w or more purity, substantially free ofinterfering proteins and contaminants. Preferably, the polypeptides areisolated or synthesized in a purity of at least 80% w/w, and morepreferably, in at least about 95% w/w purity. Using conventional proteinpurification techniques, homogeneous polypeptide compositions of atleast about 99% w/w purity can be obtained. For example, the proteinsmay be purified by use of the antibodies described hereinafter using theimmunoabsorbant affinity columns described hereinabove.

The method of the invention may be accomplished using immunoassaytechniques well known to those of skill in the art, including, but notlimited to, using microplates and lateral flow devices. In oneembodiment, an antibody specific for NT-proBNP protein is immobilized ona solid support at a distinct location. Following addition of thesample, detection of protein-antibody complexes on the solid support canbe by any means known in the art. For example, U.S. Pat. No. 5,726,010,which is incorporated herein by reference in its entirety, describes anexample of a lateral flow device, the SNAP® immunoassay device (IDEXXLaboratories), useful in the present invention. In another aspect, thesolid support is a well of a microtiter plate.

Immobilization of one or more analyte capture reagents, e.g., antibodiesto NT-proBNP, onto a device or solid support is performed so that ananalyte capture reagent will not be washed away by the sample, diluentand/or wash procedures. One or more analyte capture reagents can beattached to a surface by physical adsorption (i.e., without the use ofchemical linkers) or by chemical binding (i.e., with the use of chemicallinkers). Chemical binding can generate stronger attachment of specificbinding substances on a surface and provide defined orientation andconformation of the surface-bound molecules.

In another aspect, the invention includes one or more labeled specificbinding reagents that can be mixed with a test sample prior toapplication to a device for of the invention. In this case it is notnecessary to have labeled specific binding reagents deposited and driedon a specific binding reagent pad in the device. A labeled specificbinding reagent, whether added to a test sample or pre-deposited on thedevice, can be for example, a labeled NT-proBNP monoclonal antibody.

The detection method may include the use of a standard such as arecombinant NT-proBNP polypeptide. The standard can be mixed with themonoclonal antibody or antibodies in the same manner as the sample. Theamount of binding between the monoclonal antibody or antibodies and thestandard can be compared to the amount of binding of the antibodies tothe protein in the sample. Accordingly, because the amount of NT-proBNPin the standard is known, the amount of protein in the sample can bedetermined.

Any or all of the above embodiments can be provided as a kit. In oneparticular example, such a kit would include a device complete withspecific binding reagents (e.g., a non-immobilized labeled specificbinding reagent and an immobilized analyte capture reagent) and washreagent, as well as detector reagent and positive and negative controlreagents, if desired or appropriate. In addition, other additives can beincluded, such as stabilizers, buffers, and the like. The relativeamounts of the various reagents can be varied, to provide forconcentrations in solution of the reagents that substantially optimizethe sensitivity of the assay. Particularly, the reagents can be providedas dry powders, usually lyophilized, which on dissolution will providefor a reagent solution having the appropriate concentrations forcombining with a sample.

The device may also include a liquid reagent that transports unboundmaterial (e.g., unreacted fluid sample and unbound specific bindingreagents) away from the reaction zone (solid phase). A liquid reagentcan be a wash reagent and serve only to remove unbound material from thereaction zone, or it can include a detector reagent and serve to bothremove unbound material and facilitate analyte detection. For example,in the case of a specific binding reagent conjugated to an enzyme, thedetector reagent includes a substrate that produces a detectable signalupon reaction with the enzyme-antibody conjugate at the reactive zone.In the case of a labeled specific binding reagent conjugated to aradioactive, fluorescent, or light-absorbing molecule, the detectorreagent acts merely as a wash solution facilitating detection of complexformation at the reactive zone by washing away unbound labeled reagent.

Two or more liquid reagents can be present in a device, for example, adevice can comprise a liquid reagent that acts as a wash reagent and aliquid reagent that acts as a detector reagent and facilitates analytedetection.

A liquid reagent can further include a limited quantity of an“inhibitor”, i.e., a substance that blocks the development of thedetectable end product. A limited quantity is an amount of inhibitorsufficient to block end product development until most or all excess,unbound material is transported away from the second region, at whichtime detectable end product is produced.

Detection of NT-proBNP by Mass Spectroscopy

Peptides and peptide fragments of the invention may be detected usingstandard mass spectrometric techniques. In mass spectrometry (MS), theanalyte is first vaporized and ionized, then the ions are directed to amass analyzer, in which electromagnetic fields are used to separatespecies by mass. Lastly, a detector quantifies the abundance of eachspecies.

Standard ionization techniques that are well known in the art includeelectrospray ionization (ESI) and matrix-assisted laser desorptionionization (MALDI), which are commonly used with liquid and solidbiological samples. Other ionization technologies known in the artinclude thermal ionization (TIMS), spark ionization, secondary ion massspectrometry (SIMS), atmospheric pressure chemical ionization (APCI),ion attachment ionization, direct analysis in real time (DART),desorption/ionization on silicon (DIOS), thermospray ionization, fastatom bombardment (FAB), field desorption, glow discharge, andinductively couple plasma (ICP). Examples of well-known mass analyzersinclude sector field, time-of-flight (TOF), quadrupole, quadrupole iontrap (IT), linear quadrupole ion trap, Fourier transform ion cyclotronresonance (FT-ICR), and orbitrap mass analyzers. Examples of well-knowndetectors include electron multiplier, Faraday cup, microchannel plate,and Daly detectors.

Mass analyzers segregate ions according to their mass-to-charge ratio(m/z, where m is mass and z is charge). For example, a peptide ofmolecular weight P with two positive charges will have a net mass of(P+2) due to the ionizing presence of two protons, and will appear on amass spectrum at m/z=(P+2)/2. Thus, in general, a molecule of molecularweight M with charge Z will appear on a mass spectrum at m/z=(M+Z)/Z.

Mass spectrometry can be combined with liquid chromatography (LC) or gaschromatography (GC) to facilitate detection and identification ofcompounds residing within a complex mixture. In liquidchromatography-mass spectrometry (LC/MS), the analyte solution is firstpassed through a high-performance liquid chromatography (HPLC) column,which may separate components by a characteristic such ashydrophobicity, partition coefficient, polarity, bioaffinity, charge, orsize using well-known chromatographic techniques such as reversed-phasechromatography, normal phase chromatography, displacementchromatography, partition chromatography, ion exchange chromatography,size exclusion chromatography, or bioaffinity chromatography. The massspectrometer then generates a mass spectrum for each peak in the HPLCchromatogram. Using LC/MS, a single species of interest can be separatedand identified from within complex biological mixtures such as plasma orserum.

LC can also be combined with a tandem mass spectrometer (MS/MS), whichis capable of multiple rounds of mass spectrometry for purposes such asprotein identification. An MS/MS system can isolate and stabilize anindividual ion species appearing in a mass spectrum, which is thenfragmented using any one of several well-known techniques, such ascollision-induced dissociation (CID), electron capture dissociation(ECD), electron transfer dissociation (ETD), infrared multiphotondissociation (IRMPD), and blackbody infrared radiative dissociation(BIRD). The fragments are themselves analyzed by MS to generate afragment mass spectrum, also called a fragmentation pattern. In the caseof protein identification, an experimentally derived fragmentationpattern (generated from a sample with an unknown protein or peptide) canbe compared with fragmentation patterns that have been predicted frominformation in protein sequence or nucleotide sequence databases. A highdegree of similarity between experimental and predicted fragmentationpatterns leads to identification of the unknown protein in the sample.

Other features and advantages of the invention will be apparent from thefollowing Examples. The following are provided for exemplificationpurposes only and are not intended to limit the scope of the inventiondescribed in broad terms above. All references cited in this disclosureare incorporated herein by reference.

EXAMPLES

LC/MS analyses were obtained using a Thermo-Scientific LTQ ORBITRAPDISCOVERY™ system.

For plasma extraction, 100 uL of plasma was precipitated by addition of200 uL methanol in a 1.5 mL protein LoBind eppendorf tube. Aftervortexing for 10 seconds, the precipitate was pelleted by centrifugationat 13,000 rpm for 15 minutes at 10° C. using a benchtop centrifuge. Thesupernatant was transferred to a limited volume glass HPLC vial andanalyzed by LC/MS as described in the following examples.

Example 1 Mass Spectra of rNT-proBNP in Water, Serum Extract, and PlasmaExtract.

A sample containing canine rNT-proBNP in water at a concentration of 10μg/mL was analyzed by LC/MS. The resulting mass spectrum contains apredominant peak corresponding to the +10 charge state of the NT-proBNPpeptide (m/z=821.9).

To demonstrate detection of the peptide in serum and plasma extracts, 1μg/mL of NT-proBNP was added to water, serum extract, and plasmaextract, and each sample was then analyzed by LC/MS Full Scan mode. The+10 charge state of NT-proBNP (m/z=821.9) is clearly visible in allresulting spectra.

An additional experiment was conducted to compare mass spectra of serumand plasma extracts with and without the addition of 1 μg/mL of caninerNT-proBNP. As above, each sample was analyzed by LC/MS full scan mode.Peaks corresponding to rNT-proBNP are clearly visible in samples withrNT-proBNP added, and are absent from the blank samples (withoutNT-proBNP added).

Example 2 MS Analysis of Degradation Kinetics of NT-proBNP in CaninePlasma

Degradation kinetics of whole rNT-proBNP in canine plasma wereinvestigated by adding 1 μg to 1 mL of canine plasma; then, aliquots ofthe plasma were analyzed by LC/MS at 0, 1, 5, 24, and 48 hours afteraddition of the peptide. As shown in FIG. 2, approximately 80% of thepeptide was degraded after 24 hours.

The LC gradient is shown in Table 3, where Solvent C is 1% acetic acidin water and Solvent D is 1% acetic acid in acetonitrile.

TABLE 3 LC gradient for analysis of degradation products. No Time A % B% C % D % μL/min 1 0 0 0 95 5 500 2 1.5 0 0 95 5 500 3 11.5 0 0 5 95 5004 14.5 0 0 5 95 500 5 16.0 0 0 95 5 500 6 17.5 0 0 95 5 500

-   -   Column: Acquity UPLC BEH300 C4 1.7 μM 2.1 id×50 mm length    -   Guard Column: vanguard BEH 300 C4 1.7 uM    -   Injection volume: 20 μL    -   Tray temp: 20° C.    -   Column oven temp: 30° C.    -   MS run time: 17.5 minutes

The divert valve parameters used for degradation product LC/MS analysisare shown in Table 4.

TABLE 4 Divert Time Valve State 0.00 To waste 2.07 To source 15.57 Towaste

MS scan event 1: FTMS; resolution 30000; scan range 700.0-1800.0

MS scan event 2: FTMS; resolution 30000; scan range 821.4-822.4

MS Tune File Values

-   -   Source Type: ESICapillary Temp (° C.): 350.00    -   Sheath gas Flow: 34.5    -   Aux Gas Flow: 2.0    -   Sweep Gas Flow: 0    -   FTMS Injection waveforms: off    -   FTMS AGC Target: 500000    -   Source voltage (kV): 4.00    -   Source current (μA): 100.00    -   Capillary Voltage (V): 39.00    -   Tube Lens (V): 110.00    -   Skimmer Offset (V): 0.00    -   Multipole RF Amplifier (Vp-p): 400.00    -   Multipole 00 offset (V): −3.75    -   Lens 0 Voltage (V): −3.50    -   Multipole 0 offset (V): −6.00    -   Lens 1 Voltage (V): −23.00    -   Gate Lens offset (V): −22.00    -   Multipole 1 offset (V): −6.50    -   Front Lens (V): −6.50    -   FTMS full microscans: 3    -   FTMS full Max Ion Time (ms): 100

Example 3 Characterization of Trypsin and Proteinase K DegradationProducts by MS

To characterize the proteolytic degradation products of caninerNT-proBNP, 2 μg of proteomics grade trypsin (Sigma Aldrich, St. LouisMo.) was added to 1 μg of NT-proBNP in 1 mL of 50 mM Tris acetatebuffer, pH 8.5. The mixture was incubated at 37° C. overnight, thenanalyzed by LC/MS. Ethanol was injected as a blank.

LC/MS of degradation products was conducted using the followingparameters.

The LC gradient is shown in Table 5, where Solvent C is 1% acetic acidin water and Solvent D is 1% acetic acid in acetonitrile.

TABLE 5 No Time A % B % C % D % μL/min 1 0 0 0 100 0 300 2 5.0 0 0 100 0300 3 65.0 0 0 70 30 300 4 68.0 0 0 5 95 300 5 78.0 0 0 5 95 300 6 80 00 100 0 300 7 90 0 0 100 0 300

-   -   Column: Acquity UPLC BEH300 C4 1.7 μM 2.1 id×150 mm length    -   Guard Column: vanguard BEH 300 C4 1.7 uM    -   Injection volume: 20 μL    -   Tray temp: 20° C.    -   Column oven temp: 30° C.    -   MS run time: 90.0 minutes

The divert valve parameters used for stable peptide LC/MS analysis areshown in Table 6.

TABLE 6 DivertTime Valve State 0.00 To waste 5.05 To source 84.25 Towaste

MS scan event 1: FTMS; resolution 30000; scan range 150.0-1500.0

MS scan event 2-6: ITMS+c norm Dep MS/MS 1^(st), 2^(nd), 3^(rd), 4^(th),and 5^(th); most intense ion from respectively from scan 1.

-   -   Activation Type: CID    -   Min Signal Required: 1000    -   Isolation Width: 2.0    -   Normalized Coll. Energy: 35.0    -   Default Charge State: 2    -   Activation Q: 0.250    -   Activation Time: 30.000    -   CV=0.0V

Data Dependent Settings:

-   -   Use separate polarity settings disabled    -   Parent Mass List: none    -   Reject Mass List: none    -   Neutral loss Mass List: none    -   Product Mass List: none    -   Neutral loss in top: 3    -   Product in top: 3    -   Most intense if no parent masses found not enabled    -   Add/subtract mass not enabled    -   FT master scan preview mode enabled    -   Charge state screening enabled    -   Monoisotopic precursor selection enabled    -   Non-peptide monoisotopic recognition not enabled    -   Charge state rejection enabled    -   Unassigned charge states: rejected    -   Charge state 1: not rejected    -   Charge state 2: not rejected    -   Charge state 3: not rejected    -   Charge state 4+: not rejected

Global Data Dependent Settings

-   -   Use global parent and reject mass lists not enabled    -   Exclude parent mass from data dependent selection not enabled    -   Exclusion mass width relative to mass    -   Exclusion mass width relative to low (ppm): 20.00    -   Exclusion mass width relative to high (ppm): 20.00    -   Parent mass width by mass    -   Parent mass width low: 0.500000    -   Parent mass width high: 0.500000    -   Reject mass width relative to mass    -   Reject mass width low (ppm): 20.00    -   Reject mass width high (ppm): 20.00    -   Zoom/UltraZoom scan mass width by mass    -   Zoom/UltraZoom scan mass low: 5.00    -   Zoom/UltraZoom scan mass high: 5.00    -   FT SIM scan mass width low: 5.00    -   FT SIM scan mass width high: 5.00    -   Neutral Loss candidates processed by decreasing intensity    -   Neutral loss mass width by mass    -   Neutral Loss mass width low: 0.50000    -   Neutral Loss mass width high: 0.50000    -   Product candidates processed by decreasing intensity    -   Product mass width by mass    -   Product mass width low: 0.50000    -   Product mass width high: 0.50000    -   MS mass range: 0.00-1000000.00    -   Use m/z values as masses not enabled    -   Analog UV data dep. Not enabled    -   Dynamic exclusion enabled    -   Repeat Count: 1    -   Repeat Duration: 30.00    -   Exclusion List Size: 500    -   Exclusion Duration: 60.00    -   Exclusion mass width relative to mass    -   Exclusion mass width low (ppm): 20.00    -   Exclusion mass width high (ppm): 20.00    -   Isotopic data dependence not enabled    -   Mass Tags data dependence not enabled    -   Custom Data Dependent Settings not enabled

MS Tune File Values

-   -   Source Type: ESI    -   Capillary Temp (° C.): 350.00    -   Sheath gas Flow: 34.5    -   Aux Gas Flow: 2.0    -   Sweep Gas Flow: 0    -   FTMS Injection waveforms: off    -   FTMS AGC Target: 500000    -   Source voltage (kV): 4.00    -   Source current (μA): 100.00    -   Capillary Voltage (V): 33.00    -   Tube Lens (V): 235.00    -   Skimmer Offset (V): 0.00    -   Multipole RF Amplifier (Vp-p): 400.00    -   Multipole 00 offset (V): −3.25    -   Lens 0 Voltage (V): −4.00    -   Multipole 0 offset (V): −6.00    -   Lens 1 Voltage (V): −12.00    -   Gate Lens offset (V): −58.00    -   Multipole 1 offset (V): −6.50    -   Front Lens (V): −6.50    -   FTMS full microscans: 3    -   FTMS full Max Ion Time (ms): 100

The predominant Tryptic fragments observed were GVLAPHDSVLQALR (SED IDNO: 9) (mol. wt. 1475), SHSPAEAPEAGGTPR (SED ID NO: 8) (mol. wt. 1463),LKDAVSELQAEQLALEPLHR (SED ID NO: 36) (mol. wt. 2260), andSPASEASEASEASGLWAVQELLGR (SED ID NO: 6) (mol. wt. 2445), which werehighly similar to the fragments predicted using Peptide Cutterapplication available at the ExPASy (Expert Protein Analysis System)proteomics server of the Swiss Institute of Bioinformatics (SIB) (seeTable 1 for theoretical tryptic map of NT-proBNP).

A similar experiment was conducted using proteinase K instead oftrypsin. 1 μg of proteomics grade proteinase K was added to 10 μg ofcanine rNT-proBNP in 1 mL of 50 mM Tris HCl and 10 mM CaCl₂, pH 8.0. Themixture was incubated at 47° C. for 3 hours, then analyzed by LC/MS.Ethanol was injected as a blank. ALEPL (SED ID NO: 28) (mol. wt. 542),HPLGGRSPASEAS (SED ID NO: 40) (mol. wt. 1265), DAVSEL (SED ID NO: 41)(mol. wt. 633), and LEPL (SED ID NO: 42) (mol. wt. 471) were among theproteinase K fragments observed in the mass spectrum. A theoreticalproteinase K digestion map is shown in Table 2.

Example 4 Characterization of Stable Degradation Products of CanineNT-proBNP

To characterize the rNT-proBNP stable degradation products present afterincubation in canine plasma, canine rNT-proBNP was added to canineplasma at 1, 5, and 10 μg/mL and incubated at room temperature. At 24,48, 96, and 192 hours, 100 μL aliquots of the plasma were collected andcombined with 200 μL of methanol to form a precipitate. Aftercentrifugation, the supernatant was analyzed for stable peptides byLC/MS as described in Example 3.

After 24 hours, the fragment LGGRSPASEASEASEASGLWAVQELLGR (SED ID NO:14) was observed in the sample containing 10 μg/mL NT-proBNP, indicatingcleavage between the detector and capture regions of the full-lengthpeptide.

After 192 hours, identified fragments included HSPAEAPEAGGTPRVLAPHDSVLQ(SED ID NO: 30), GVLAPHDSVLQ (SED ID NO: 31), andLWAVQELLGRLKDAVSELQAEQLALEPLHRSHSPAEAPEAGGTPRGVL (SED) ID NO: 24).

The relative abundance of several fragments from the N-terminal,capture, and C-terminal regions were evaluated over time. These data areshown in FIGS. 3-6, and indicate that the most stable fragment sequencesover time were GRSPASEASEASEASGLWAVQ (SEQ ID NO: 22) from the N-terminalregion and SHSPAEAPEAGGTPRGVLAPHDSVLQ (SEQ ID NO: 30) from theC-terminal region.

Example 5 Identification of Stable Epitopes of Canine NT-proBNP

To identify stable peptide epitopes, a database containing the aminoacid sequence of rNT-proBNP HPLGGRSPAS EASEASEASG LWAVQELLGRLKDAVSELQAEQLALEPLHR SHSPAEAPEA GGTPRGVLAP HDSVLQALR (SEQ ID NO: 4) inFASTA format was created. The .RAW file of interest acquired from theLC-MS run was uploaded along with the FASTA database to ProteomeDiscoverer software (v 1.0 Thermo Scientific) for identification of thestable epitopes.

The peptides that satisfy the criteria as directed in the ProteomeDiscover Software were indentified as stable epitopes for a particulartime series.

Example 6 Immunoassay

Immunoassays were conducted using monoclonal antibodies specific for SEQID stable canine NT-proBNP degradation fragments (SEQ ID NOS: 22 and 30)at 24, 48, 72 and 96 hours. Sheep polyclonal antibodies raised againstc-KDAVSELQAEQLALEPL (SEQ ID NO: 37) were coated on a solid phase.Detection antibodies were monoclonal antibodies raised against the SEQID NOS: 22 and 30 conjugated at both the N and C terminus with a proteincarrier: (i) GRSPASEASEASEASGLWAVQ-(PEG)6-Cys-KLH (SEQ ID No. 32) andKLH-Cys-(PEG) 6-GRSPASEASEASEASGLWAVQ (SEQ ID No. 33); and/or ii)SHSPAEAPEAGGTPRGVLAPHDSVLQ-(PEG)6-Cys-KLH (SEQ ID No. 34) andKLH-Cys(PEG)6-SHSPAEAPEAGGTPRGVLAPHDSVLQ (SEQ ID No. 35). The monoclonaldetection antibodies raised against the above immunogens were labeledwith HRP using standard techniques. Canine plasma was incubated with thesolid phase for 1 hour followed by a second incubation with the labeleddetection antibodies. Results of the assay are shown in FIGS. 7 and 8.In this method, the assay detected longer fragments that included bothSEQ ID NO: 37 and one of SEQ ID NO: 22 and 30.

The examples given above are merely illustrative and are not meant to bean exhaustive list of all possible embodiments, applications ormodifications of the invention. Thus, various modifications andvariations of the described methods and systems of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific embodiments, it should be understood thatthe invention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, immunology, chemistry, biochemistry or in therelevant fields are intended to be within the scope of the appendedclaims.

It is understood that the invention is not limited to the particularmethodology, protocols, and reagents, etc., described herein, as thesemay vary as the skilled artisan will recognize. It is also to beunderstood that the terminology used herein is used for the purpose ofdescribing particular embodiments only, and is not intended to limit thescope of the invention.

The embodiments of the invention and the various features andadvantageous details thereof are explained more fully with reference tothe non-limiting embodiments and/or illustrated in the accompanyingdrawings and detailed in the following description. It should be notedthat the features illustrated in the drawings are not necessarily drawnto scale, and features of one embodiment may be employed with otherembodiments as the skilled artisan would recognize, even if notexplicitly stated herein.

Any numerical values recited herein include all values from the lowervalue to the upper value in increments of one unit provided that thereis a separation of at least two units between any lower value and anyhigher value. As an example, if it is stated that the concentration of acomponent or value of a process variable such as, for example, size,angle size, pressure, time and the like, is, for example, from 1 to 90,specifically from 20 to 80, more specifically from 30 to 70, it isintended that values such as 15 to 85, 22 to 68, 43 to 51, 30 to 32,etc. are expressly enumerated in this specification. For values whichare less than one, one unit is considered to be 0.0001, 0.001, 0.01 or0.1 as appropriate. These are only examples of what is specificallyintended and all possible combinations of numerical values between thelowest value and the highest value enumerated are to be considered to beexpressly stated in this application in a similar manner.

Particular methods, devices, and materials are described, although anymethods and materials similar or equivalent to those described hereincan be used in the practice or testing of the invention. The disclosuresof all references and publications cited herein are expresslyincorporated by reference in their entireties to the same extent as ifeach were incorporated by reference individually.

What is claimed is:
 1. A method for determining an amount of N-terminal pro-Brain Natriuretic Peptide (NT-proBNP) in a sample from a canine, comprising determining an amount of a polypeptide consisting of GRSPASEASEASEASGLWAVQ (SEQ ID NO: 22) in the sample, and correlating the amount of the polypeptide in the sample to the amount of NT-proBNP in the sample.
 2. The method of claim 1, wherein the step of determining the amount of the polypeptide comprises contacting the sample with a first antibody that specifically binds a first epitope on SEQ ID NO:22.
 3. The method of claim 2, wherein the first antibody is a monoclonal antibody.
 4. The method of claim 2, wherein the step of determining the amount of the polypeptide further comprises contacting the sample with a second antibody that specifically binds a second epitope on SEQ ID NO:22, wherein the first epitope is different than the second epitope.
 5. The method of claim 4, wherein the second antibody is a monoclonal antibody.
 6. The method of claim 4, wherein the first antibody is bound to a solid phase and the second antibody is bound to a label.
 7. The method of claim 4, wherein the first antibody is bound to a label and the second antibody is bound to a solid phase.
 8. The method according to claim 1, wherein the step of determining the amount of NT-proBNP comprises mass spectroscopy.
 9. A method for determining a presence or an amount of canine N-terminal pro-Brain Natriuretic Peptide (NT-proBNP) in a biological sample comprising detecting a degradation product of NT-proBNP in the sample, wherein the degradation product consists of SEQ ID NO:
 22. 10. The method of claim 9, wherein the step of detecting the degradation product comprises contacting the sample with a first antibody that specifically binds a first epitope on SEQ ID NO:22.
 11. The method of claim 10, wherein the first antibody is a monoclonal antibody.
 12. The method of claim 10, wherein the step of detecting the degradation product comprises contacting the sample with a second antibody that specifically binds a second epitope on SEQ ID NO:22, wherein the first epitope is different than the second epitope.
 13. The method of claim 12, wherein the second antibody is a monoclonal antibody.
 14. The method of claim 12, wherein the first antibody is bound to a solid phase and the second antibody is bound to a label.
 15. The method of claim 12, wherein the first antibody is bound to a label and the second antibody is bound to a solid phase. 